长链非编码RNA Gm35082-202对布鲁氏菌引起的细胞焦亡的影响

doi:10.16431/j.cnki.1671-7236.2022.09.034

Abstract

Abstract: 【Objective】 This study was aimed to explore the effect of long non-coding RNA(lncRNA) Gm35082-20 on pyrolysis during Brucella infection, which laid a foundation for further research on the involvement of lncRNA in regulating immune response induced by Brucella.【Method】 The chromosome location and exon information of Gm35082-202 were searched and analyzed by Ensemble database, the secondary structure of Gm35082-202 was predicted though RNAfold online software, KEGG and GO enrichment analysis were used to investigate the function of Gm35082-202.RAW264.7 cells infected by Brucella at different time (0, 4, 8, 12, 24, 36 and 48 h) with MOI 0.01 were collected, and the expression of Gm35082-202 detected by Real-time quantitative PCR.The subcellular localization of Gm35082-202 was predicted by LncLocator website, and the expression of Gm35082-202 was detected by Real-time quantitative PCR in the nuclear and cytoplasmic.3 pieces siRNAs of Gm35082-202(siRNA1, siRNA2 and siRNA3) were designed and transfected to RAW264.7 cells, then the expression of Gm35082-202 was detected by Real-time quantitative PCR to screen siRNA with the highest interference efficiency for subsequent tests.After RAW264.7 cells were transinfected with Gm35082-202-siRNA and Gm35082-202-NC for 24 h, and then infected with Brucella.PBS was used as blank control group (PBS), and only Brucella infected cells were used as infection control group (Bru).Cells in PBS, Bru, Gm35082-202-siRNA (Bru-siRNA) and Gm35082-202-NC (Bru-NC) groups were collected, the mRNA and/or protein expressions of NLRP3, Caspase-1, Caspase-11, IL-1 and IL-18 were detected by Real-time quantitative PCR and Western blotting, respectively.The secretion levels of IL-1β and IL-18 in supernatant of each group were detect by ELISA.The intracellular viability of Brucella in each group was compared by colony count.【Result】 Gene structure analysis showed that Gm35082-202 was a lncRNA of 525 nt (Trianscript ID:ENSMUST00000208164.2), contained 2 exons, located in chromosome 7 of mouse(Chr7:100 324 295-100 326 967), its secondary structure contained multiple stem loops and multi-branched internal loop structures.KEGG signaling pathway enrichment analysis showed that Gm35082-202 mainly involved in regulating NOD-like receptor, calcium regulation and cytokine signaling pathway.GO function analysis showed that the function of Gm35082-202 mainly involved in DNA transcription, mitochondrial components and metal ion binding.After Brucella infected RAW264.7 cells, compared with 0 h group, the expression of Gm35082-202 was significantly increased at 4 and 8 h (P<0.05), and extremely significantly increased at 12, 24, 36 and 48 h (P<0.01).The prediction results of subcellular localization showed that Gm35082-202 was mainly distributed in nucleus (58%), followed by cytoplasm (35%).When RAW264.7 cells were infected by Brucella for 0 h, the distribution of Gm35082-202 in the nucleus was 63.4%, while the distribution of Gm35082-202 decreased to 24.5%, 29.6% and 30.4% at 4, 24 and 36 h, respectively.The expression of Gm35082-202 was extremely significantly reduced by Gm35082-202-siRNA1 and Gm35082-202-siRNA3 (P<0.01), and Gm35082-202-siRNA1 had the highest interference efficiency.Compared with Bru group, in Bru-siRNA group, the expressions of NLRP3, Caspase-1, Caspase-11, IL-1β and IL-10 genes were extremely significantly reduced(P<0.01), the expressions of NLRP3 and Caspase-11 protein were significantly decreased(P<0.05), whlie the expression of Caspase-1 protein was extremely significantly decreased(P<0.01), and the secretion of IL-1β and IL-18 in supernatant was extremely significantly decreased (P<0.01), and the intracellular Brucella count was increased significantly (P<0.05).【Conclusion】 Brucella promoted pyroapoptosis of macrophages by up-regulating Gm35082-202 expression, which could provide references for further exploring the molecular mechanism of Gm35082-202 in regulating pyroapoptosis of host cells during Brucella infection.

Key words: Brucella; Gm35082-202; pyroptosis

CLC Number:

S852.61⁺4

DENG Xingmei, CAO Shuzhu, GUO Jia, ZHU Dexin, ZHAO Tianyi, CHAI Yingjin, ZHANG Wei, SHI Chao, JIA Sifeng, ZHANG Hui. Effect of Long Non-coding RNA Gm35082-202 on Cell Pyrodecay Induced by Brucella[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3599-3609.

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Effect of Long Non-coding RNA Gm35082-202 on Cell Pyrodecay Induced by Brucella

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